Ionic and electrical gradients across cell and organelle membranes are created by pumps, whose correct functioning is essential to all biological and biomedical processes. Dysfunctional membrane proteins of this kind underlie many cardiac, neurological and digestive diseases, and they have been prominent targets of pharmaceutical intervention. We will study and describe proton transfer mechanisms in a newly discovered family of proton pumps that deviate from those in a model system employed earlier to generate general principles for active ion transport. The aims of this proposal focus on the functional implications of our recently reported x-ray diffraction structure of xanthorhodopsin. We expect to uncover novel means of proton transfer in proteins, with insights into how energy input is used to move protons uphill, across the membrane. We hope that the findings will enrich the general field of membrane proteins, and that of transport proteins in particular. We will explore the consequences of three main differences from the x-ray structure of bacteriorhodopsin that we had reported earlier. a) The critical proton transfer, which in these proteins drives the transport, is from the retinal Schiff base to an aspartate connected via a water molecule at the active site. We now know that the proton acceptor can be also an aspartate-histidine complex with very likely shared protons. b) Release of protons to the bulk can be via an extended hydrogen-bonded network of polar residues and bound water, but now we know that an alternative exists that utilizes a deep cleft that extends into the proteins. c) Uptake of protons and reprotonation of the Schiff base by an internal acidic residue is via a transient chain of water molecules that spans an otherwise hydrophobic domain, during the transport cycle. We now know that this domain may contain water molecules that connect the proton donor to the Schiff base region. The experimental approaches to uncover the changed functions include the battery of methods we and our collaborators had developed over the past decades: construction of site-specific mutants expressed in E. coli, static and transient optical spectroscopy (absorption and fluorescence), site-specific spin labeling, Fourier Transform Infrared spectroscopy, crystallization and x-ray diffraction, and solution NMR. PUBLIC HEALTH RELEVANCE: Life and health depend on maintaining the correct ionic balance between the interior of cells and their surroundings. The proposed work will contribute to the fundamental understanding of how pumps transport ions like protons across cell membranes. It will take advantage of novel features in a newly discovered family of pumps, and extend the range of possible mechanistic elements that add up to the translocation of the ion in these membrane devices.